Methods and compositions for chromatography

ABSTRACT

The present invention is directed to methods and compositions for separating and isolating target molecules. In particular, the present invention comprises devices, such as CCDs, that contain particles without the need for support structures. Chromatography separation techniques, including but not limited to, ion exchange, size separation, affinity chromatography, ion exclusion, ligand exchange, reversed phase and normal phase partitioning, are used in the CCD. Methods also include low, medium and high pressure liquid chromatography. Such methods can be used for analytical, semi-preparative processes, initial clarification, preparative filtration and process scale applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of Ser. No. 11/214,144, filed Aug. 29,2005, now U.S. Pat. No. 7,347,943, which, in turn, is a continuation ofSer. No. 10/293,916, filed Nov. 12, 2002, now U.S. Pat. No. 6,942,804,which, in turn, claims priority to and incorporates by reference in itsentirety, U.S. Provisional No. 60/344,745 filed Nov. 9, 2001.

TECHNICAL FIELD

This application relates to methods and devices for chromatography andcomposition used therewith.

BACKGROUND OF THE INVENTION

The goal of chromatography is to separate materials. Chromatographytechniques are used for a variety of purposes, from research in basicscience to purification of pharmaceuticals. The application ofchromatography techniques to production levels works to some degree butwhen methods and devices are scaled up to the large sizes needed forpharmaceutical or biological product production, most methods anddevices work inadequately.

There are many different kinds of chromatography methods and materials.Some of these include paper and thin layer chromatography methods,columns and resins of all types, high pressure liquid chromatography,expanded bed techniques, and reverse phase and reverse flow methods. Forexample, the first stage in many purification processes of proteins froma fermentation broth, whether from microbial, plant, or animal cellculture, is capture of the desired proteins from the broth. A typicalmethod for accomplishing this is to use an adsorbent material in anexpanded bed. On a large scale, the expanded bed uses an upwardoperating flow through the bed and the flow rate is restricted byincreased viscosity, the density of chromatographic adsorbents used, andthe rate of binding of the desired protein to the adsorbent. There arealso only a few adsorbent materials, such as beads, that can be used dueto the presence of only a few types of functional binding groups,particle size, and density of the particle. Additionally, the columnsused to perform the chromatography often become contaminated bybacterial or fungal growth, or blocked due to cellular debris. All ofthese problems lead to a slower process with less material isolated.

The majority of processes for producing pharmaceutical or diagnosticproducts involve the purification of proteins and peptides frombacteria, yeast and plant or animal cell culture fluids, or extractsfrom tissues. Usually purification plants use multiple unit operations,including a number of chromatographic steps to ensure the removal ofimpurities and contaminants. The type of product produced and itsintended use will dictate the extent of purification needed. Each stepin the recovery process will affect the overall process efficiency byincreasing operational costs and process time, and by also causing lossin product yield. Careful selection and combination of suitable unitoperations during the design phase may reduce the number of stepsneeded. The fewest possible processing steps offers the most efficientway of reaching high process efficiency and low costs in the overallproduction process. Most currently used processes still involve multiplesteps of processing which add to the costs, loss of product and offeropportunities for contamination.

Problems in isolation of materials begins in the earliest stages, suchas clarification of a fermentation broth or an initial tissuehomogenization. Standard techniques for removal of cells or debris arecentrifugation and microfiltration. The efficiency of a centrifugationstep depends on particle size, density difference between the particlesand the surrounding liquid, and viscosity of the feedstock. Althoughmicrofiltration may yield cell free solutions, the flux of liquid perunit membrane area is often dramatically decreased during the filtrationprocess. Fouling of the microfiltration membranes is another problemthat significantly adds to the operational cost. The combined use ofcentrifugation and filtration often results in long process times or theuse of comparatively large units causing significant capital expenditureand recurrent costs for equipment maintenance. It also results insignificant product loss due to product degradation. What is needed aremethods, compositions, and devices that allow for direct adsorption fromcrude feed stocks that can reduce the time and cost of the initial stepsof purification.

An alternative to methods of clarification and packed bed chromatographyis adsorption to a resin in a stirred tank. This technique is oftenuseful when recovering the target substance from a large volume of crudefeed. This method has long been used on a commercial scale for theisolation of plasma coagulation Factor IX with DEAE Sephadex. A majordrawback to this system is that well-mixed batch adsorption process is asingle-stage adsorption procedure and requires more adsorbent to achievethe same degree of adsorption as in a multi-stage (multi-plate) processsuch as packed bed chromatography.

A very widely used technique for bulk separation is adsorption of thetarget molecules in a fluidized bed. This technique can eliminate theneed for particulate removal. Fluidized beds have been used in industryfor many years for the recovery of antibiotics includingbatch-processing techniques for recovery of streptomycin andsemi-continuous systems for novobiocin. In a fluidized bed, channeling,turbulence, and backmixing is extensive, and is similar to a batchprocess in a stirred tank. The single equilibrium stage in a fluidizedbed decreases the efficiency of the adsorption process with lowrecoveries, causes the need for re-cycling the media, inefficientwashing procedures and increased processing time.

Approaches to solving these problems have been tried by many techniquesso that a fluidized bed would have separation characteristics similar topacked bed chromatography. One approach uses segmentation of the bed byinsertion of a number of plates with suitably sized holes into theadsorption column. In another approach, magnetic adsorbent particles anda magnetic field over the fluidized bed column are used to stabilize thebed. A substantial stabilization of the bed was achieved using magneticadsorbents but the experiments were carried out at small laboratoryscale and scaling up requires complicated and expensive equipment.Another approach uses agarose in a column equipped with a liquiddistribution inlet giving a plug flow in the column.

When these expanded beds were actually used with mixtures of proteinsand cells there was some improvement. The breakthrough capacity in suchbeds, expanded by a factor of two, was very similar to the breakthroughcapacity in a packed bed. However, low flow velocities had to be appliedto prevent the bed from expanding too much, which resulted in a lowoverall productivity.

Problems also occur with the particles used for separation. Manystandardly used particles are not sturdy enough to withstand the weightof a large column bed, nor can they withstand harsh chemical treatmentsused for cleaning the beds and columns. The packing materials or resinsdeteriorate over time due to clean-in-place procedures, harsh bufferconditions, and changing buffer conditions. Additionally, the entirecolumn, piping, or resins may become contaminated, either throughbacterial or fungal growth, or through accumulation of material on theparticles or resins and that lowers the efficiency. This requiresexpenditures for replacement of the resins, cleaning all equipment andthen assurances that the column has been returned to a good, reliableworking condition.

Therefore, a need exists for systems, methods, and devices that canseparate biomaterials or chemicals that overcome the problems seen withcurrently used chromatography devices. It is preferred that suchsystems, methods, and devices be capable of using chromatographicaltechniques and resins or materials to isolate and separate biomaterialsare chemicals more efficiently, and with lower production costs.

SUMMARY OF THE INVENTION

The present invention is directed to methods and devices for usingcompositions for separating and isolating biomaterials, chemicals orother materials. In particular, the present invention comprises devicesthat can contain particles without the need for support structures.Preferred methods and devices are described in U.S. Pat. Nos. 5,622,819;5,821,116; 6,133,019; and 6,214,617; and U.S. patent application Ser.Nos. 09/316,566; 09/870,928; 09/773,027; 09/788,991; and 10/153,161; allof which are incorporated herein by reference in their entireties. Ingeneral, such devices when used for growth of cells are referred to as“CBR” or centrifugal bioreactor. When applied to the separation andisolation techniques taught herein, the devices are collectivelydesignated as “CCD” or centrifugal chromatography devices.

In general, the devices of the present invention comprise novelapparatuses for containing chromatography materials, such as bedmaterials, beads, resins or gels which are immobilized within chambersmounted in a centrifugal field while liquids, with or without any gasphase(s) in contact with the liquids, are flowed into and out of thechambers. The bed materials are ordered into a three-dimensional arrayof particles, the density of which is determined by the particle size,shape, intrinsic density, and by the selection of combinations ofcontrollable parameters such as liquid flow rate and angular velocity ofrotation.

In an alternative embodiment, the bed materials are not confined inclosed chambers, but rather are immobilized in open chambers formed byand between adjacent disks. As with the other disclosed embodiments ofthis invention, the inflow of nutrient fluid into the chamber is oneforce that counterbalances the centrifugal force exerted on the bedmaterials to immobilize the bed materials in the open chamber. Using asummation of vector forces, a CCD is capable of maintaining particles ora resin in a chamber to form a bed with which materials are separatedusing chromatography techniques. All standard chromatography techniques,including but not limited to, adsorption, ion exchange, size separation,affinity chromatography, ion exclusion, ligand exchange, reversed phaseand normal phase partitioning, are used in the CCD. The chromatographymaterials used in a CCD include those materials used in chromatographymethods, and are not limited by particle fragility due to column weightconsiderations. Methods also include low, medium and high pressureliquid chromatography. Such methods can be used for analytical,semi-preparative processes, initial clarification, preparativefiltration and process scale applications. The chromatography system iseasily created in a CCD and thus, dismantling the system for cleaning,if necessary, is also easily accomplished.

In general, the methods comprise addition of a desired chromatographymaterial to the chamber or chambers of one or more CCD and forming a bedor chromatography plates by running the CCD, adding the liquid fromwhich the target molecules are to be separated, and collecting theisolated target molecules.

Accordingly, the present invention comprises methods for isolatingmaterials comprising devices comprising one or more CCDs which usecompositions for chromatography.

The present invention may be understood more readily by reference to thefollowing detailed description included herein. Although the presentinvention has been described with reference to specific details ofcertain embodiments thereof, it is not intended that such details shouldbe regarded as limitations upon the scope of the invention. The entiretext of the references mentioned herein are hereby incorporated in theirentireties by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the process of an apparatus of this invention.

FIG. 2 is an illustration of the mathematics governing the motion of aparticle due to the effect of gravity on that particle when it isrestrained in a centrifugal field that is opposed by a liquid flow.

FIG. 3 is an illustration of the resultant motion of a particle underthe constraints of FIG. 2.

FIG. 4 is a mathematical evaluation of the immobilization conditions ata given radius.

FIG. 5 is an analysis of the balance of centrifugal forces and flowvelocity forces in a rotating cylindrical bioreactor chamber.

FIG. 6 is an analysis of the balance of centrifugal forces and flowvelocity forces in a rotating conical biocatalyst immobilizationchamber.

FIG. 7 is an illustration of a three-dimensional array of particles in arotating conical biocatalyst immobilization chamber.

FIG. 8 is an illustration of the inter-stratum buffer regions in athree-dimensional array of particles in a rotating conical biocatalystimmobilization chamber.

FIG. 9 is a mathematical analysis of the intra-stratum flow velocityvariation in a two-dimensional array of particles in a rotating conicalbiocatalyst immobilization chamber.

FIG. 10 is an illustration of an example a conical-shaped immobilizationchamber and the boundary conditions which determine those dimensions.

FIG. 11 is an analysis of the positional variation of the centrifugaland flow velocity forces in the chamber of FIG. 10 at a flow rate of 10mL/min.

FIG. 12 illustrates a system according to various embodiments of theinvention.

FIG. 13 illustrates another system according to various embodiments ofthe invention.

FIG. 14 illustrates yet another system according to various embodimentsof the invention.

FIG. 15 is a front or side view of a CCD according various embodimentsof the invention.

FIGS. 16A and 16B show end and side views of a chamber of the CCDaccording to the embodiment of the invention shown in FIG. 15.

FIGS. 17A and 17B show end and cross-sectional views of one side of achamber according to the embodiment of the invention shown in FIGS. 15and 16A-B.

FIGS. 18A and 18B show end and cross-sectional views of one side of achamber according to the embodiment of the invention shown in FIGS. 15and 16A-B.

FIGS. 19A and 19B show cross-sectional views of one side of the chamberaccording to the embodiment of the invention shown in FIGS. 15 through18A-B.

FIGS. 20A and 20B show end and cross-sectional views of another side ofa chamber according to the embodiment of the invention shown in FIGS. 15and 16A-B.

FIGS. 21A and 21B show side and end views of a portion of a shaft of theCCD according to the embodiment of the invention shown in FIG. 15.

FIGS. 22A and 22B show side and end views of another portion of theshaft of the CCD according to the embodiment of the invention shown inFIG. 15.

FIGS. 23A-23E show side and cross-sectional views of a manifold sleeveof the CCD according to the embodiment of the invention shown in FIG.15.

FIG. 24 shows an end view of a CCD according to various embodiments ofthe invention.

DETAILED DESCRIPTION

In general, the present invention is directed to methods for isolatingmaterials using devices described herein and compositions comprisingmedia and particles for chromatography techniques. The inventioncontemplates the isolation of one or more specific components from amore complex material, such as isolation of proteins or peptides fromfermentation broths or tissue extracts, or chemicals from chemicalreactions. Any materials that can be isolated by chromatography methodscan be isolated by the methods, devices and compositions describedherein, and the invention is not limited by the description of specificmaterials or chromatography techniques.

Methods for isolating target molecules, similar to those currently usedfor isolating molecules, comprise using one or more of the devicesdescribed herein, referred to as CCDs, centrifugal chromatographydevices. The present invention includes, but is not limited to, methodsof clarification, filtration, single-stage adsorption, batch adsorptionabsorption methods, ion exclusion chromatography, normal phasepartition, reverse phase partition, polar separations, nonpolarseparations, hydrophobic separations, hydrophilic separations, ligandexchange, ion-pairing, size exclusion and affinity chromatographymethods.

For example, the initial purification step or steps of isolating atarget molecule begins by adsorption chromatography using a conventionalbed of adsorbent positioned within a CCD. Prior to addition of astarting material, such as a fermentation broth, a tissue extract or achemical reaction mixture, the starting material is generally clarifiedor at least, large particulates are removed. Standard techniques forremoval of cells or debris are centrifugation and microfiltration. Theefficiency of a centrifugation step depends on particle size, densitydifference between the particles and the surrounding liquid, andviscosity of the feedstock. When handling small cells, such as E. coli,or cell homogenates, small particle size and high viscosity reduce theamount of material that can be used in each centrifugation and oftenmakes it difficult to obtain a completely particle-free liquid.Centrifugal methods are known to those skilled in the art and suchmethods can be used prior to adsorption or at any step whereconcentration of target molecules is needed. Alternatively,centrifugation can be replaced by methods of separation in a CCD. Thecrude starting material can be added to a CCD used in a preparativestep, where for example, a bed of size separation beads are used tohinder the movement of larger materials in the starting material whilesmaller materials flow through to the next processing step.

To obtain a solution that can be further purified by chromatography,centrifugation is usually combined with filtration methods, such asmicrofiltration. The methods of the present invention comprise use ofCCD with pretreatment or post treatment of the material bycentrifugation or filtration steps. Filtration includes filteringmethods using membranes and filters made from known materials andcomprising pore sizes comprising ranges from nanometer to micron tomillimeter to meter sized pores.

A method contemplated in the present invention is adsorption to a resin.This technique is often useful when recovering the target substance froma large volume of crude feed. Methods of single-stage adsorption andbatch adsorption are contemplated by the present invention. Resins areadded to a CCD and the crude feed, or media to be clarified, or anyother liquid is added. The CCD and resin can be used to isolate plasmacoagulation factors with DEAE Sephadex.

The CCD and methods of the present invention for single-stage adsorptionor batch adsorption are advantageous over standard chromatographymethods because the resin or other bed material can be easily added andwithdrawn from the CCD, by, for example, changing the rotation speed.Once a batch of liquid has been through the bed, the bed is removed anda new bed of the same type or a different type is added to the CCD andprocessing can continue. In other methods, a loop system can be used toreturn the liquid through the bed more than one time to assure completeremoval of target molecules from the liquid, or to saturate the bedmaterial. Once the target molecule is removed from the liquid and isadsorbed onto the bed material, the liquid is processed in a CCD orother device downstream. If the bed material has a target moleculeadsorbed onto it that is wanted, the bed material is either treatedwithin the CCD to release the bound target molecule, or the bed materialcan be removed from the CCD and treated to remove the target molecule.If the bound target molecule is not wanted, the bed material can becleaned within the CCD or removed from the CCD and cleaned or discarded.

A method for bulk separation of target molecules is adsorption of thetarget molecules in a fluidized bed. In some applications, this methodeliminates the steps for particulate removal. Fluidized beds are createdin a CCD by establishing a bed material in the CCD by the summation ofthe vector forces, so the particles of bed material remain suspended insubstantially the same location within the CCD and then flowing themedia having the target molecules through the bed material.

There are a large number particles that can be used in the methods ofthe present invention and in the CCDs for separation of target moleculesfrom the media that contains them. For example, agarose supportparticles have long been used in chromatography methods. Commerciallyavailable adsorbents based on amorphous silica have also been used.These adsorbents are denser than agarose-based adsorbents, but thesmaller bead size enables this material to expand to the same degree asbeds of agarose beads at comparable flow velocities. There is nolimitation in the present invention for the type of particles used asthe bed material. Any particle that can be used in standardchromatography methods are contemplated for use in the methods anddevices of the present invention. Particles that specialized forparticular target molecules or for particular media conditions are alsocontemplated by the present invention.

As used herein, isolation of a target molecule includes all of the stepsinvolved in the process of isolating the target molecule. For example,steps of clarifying or centrifuging stock feed broths is included inisolation of the target molecule, generally as the first step in theprocess of isolation. The processes may or may not lead to a targetmolecule that is free of the starting material, but includes any stageof purification that is reached by a particular step or process.

Methods of the present invention comprise preparation or isolation oftarget molecules by one or more CCDs. The CCDs can be used individually,in serial arrangement or in parallel arrangements, and in combinationswith other separation techniques and apparatus. One process step can beperformed in one CCD and the eluent from a first CCD can be fed into asecond or more CCDs. This system can be used, for example, for a methodof treating large amounts of liquid in CCDs, all of which comprise thesame bed material, so that all of the liquid is treated at one time bymany CCDs, in either a serial pathway or a parallel pathway.Alternatively, a method comprises adding a liquid comprising a targetmolecule to a first CCD having a particular bed material, having theliquid effected by the conditions in the CCD, and then adding the liquidleaving the first CCD to a second CCD having a different bed material.For example, the crude feed stock is added to a first CCD having a bedmaterial that provides a sizing function and removes larger materialssuch as cells or cellular debris. The liquid leaving the first CCD,having been acted on by the bed materials, no longer contains as manycells or as much cellular debris as it did prior to being exposed to thebed material. This treated liquid is then fed into a second CCD, thathas a bed material that acts as an ionic exchanger.

In these methods, one processing step can be performed on a large amountof starting material, such as stock or broth material, chemicalreactions or tissue extracts, or multiple steps of purification orisolation can be performed. Additionally, the starting material can befed into one CCD in a continuous loop in order to provide multiplepasses of the heterogeneous liquid over the isolating or separatingmaterial particles) contained within the chamber or chambers of the CCD.In this way, a particular target molecule can be thoroughly removed fromthe starting material, which leads to greater amounts of target moleculeobtained, or the liquid can be cleared of unwanted materials.

The chromatography materials added to one or more CCDs include all knowntypes of materials used in chromatography techniques, particularly thoseused in packed bed or expanded bed columns, and low, medium and highperformance liquid chromatography columns. The choice of material addedto one or more chambers of the CCDs is determined by the startingmaterial, such as tissue extract, chemical reaction mixture or stockbroth, any preprocessing steps, and the target molecule or molecules tobe isolated. In the methods of the present invention, because the CCDcontains the bed materials by a combination of vector forces and doesnot rely upon either a support structure or the other bed materials tohold the bed materials in place, compression of the bed materials doesnot occur like that found in conventional column techniques. Therefore,smaller materials or materials not capable of being used at higherpressures can be used in the present invention. The choice ofchromatography materials is not limited by the same considerations asthose found in conventional chromatography and the present inventioncontemplates such novel uses of these materials. The present inventionalso comprises methods and compositions of buffers and eluents that areknown to those skilled in the art.

A beneficial aspect of the present invention is that the CCD is easilymaintained. In general, the chromatography material, herein referred toas resin, gel, bead, or bed material, is easily added to the chamber orchambers through a port. The bed is formed by, among other forces,rotation of the chamber to the desired speed, to yield the size andshape needed for the particular application. The combination of thevectors of force, including, but not limited to, centrifugal force, theforce of the media stream and gravity, allow the particles to form a bedin the interior of the chamber of the CCD wherein each particle isindependently suspended in relation to every other particle of resin.This allows for efficient exposure to all surfaces of the particle, nopacking of the particles, no back flow pressure problems and theparticles are maintained within the chamber, so that there is nocontamination of the media exiting the chamber with particles. Bychanging the forces, including the rotation parameters, the particlescan be easily flushed from the chamber if necessary, and the emptychambers can be cleaned or sterilized for another run. The particles canbe recharged or cleaned while they are maintained within the chamber orflushed, cleaned and added back to the chamber.

The forces created in the CCD cause the bed within the chamber to formquickly after addition of the particles, which are generally added withliquid. Once the preferred force summation, including rotationparameters, is reached within the CCD, the bed will maintain the desireddensity and shape. Should processing steps require a different flowspeed of the target molecule through the bed, the density and shape ofthe bed is easily changed by changing the force parameters of the CCD.One CCD has the capability of providing a multitude of differentchromatography techniques, even without changing the particle type. Forexample, a CCD with sizing particles, at a particular force summationparameter, can be used to initially filter a fermentation broth bypassing the broth quickly through the bed so that only large materialsare retained, by for example, forming a lower density bed and havinghigher flow media so that only large materials are retained. Theparticle bed is easily cleaned by washing. The same CCD can then form amore dense particle bed by using different rotation and operating forceparameters that allow for longer retention time by the target molecule.

The CCD methods and compositions overcome the problems with packed bedchromatography, while providing excellent separation. The packed bed isa depth filter, and this it is an excellent collection device forparticulate matter. The smaller the packing media, the better it acts asa filter. Bonded resin column packing materials are suitable forseparating certain solutes, but are also capable of retaining othercomponents of the sample indefinitely. These retained compounds maysignificantly decrease column efficiency and selectivity. If proper careof the column is not taken then time and money are wasted when thecolumn is ruined in a short time. Column maintenance is a constantexpense with attendant labor costs.

The CCD does not accumulate materials due to packing constraints becausea change in the force summation parameters can expand the distancebetween each packing particle so that the each particle can be flushedclean on all surfaces. Additionally, if the packing materials becomecontaminated with adhered materials, the packing materials can be easilyflushed out of the CCD chamber and new packing material added while theoriginal packing material is cleaned or recharged, or the material maybe cleaned or recharged within the chambers. The chambers of CCD can bemade from any sturdy material and therefore are resistant to harshbuffers or materials, making them easily cleaned.

Examples are provided herein for applications of the methods andcompositions of the present invention. These examples are forillustration and are not to be seen as limiting the invention. Theinvention comprises separation of molecules using CCD, and anyseparation techniques that can be adapted to a CCD are contemplated bythe present invention. In particular, the invention comprisescompositions comprising known particle types and novel applications ofparticle types that cannot be currently used because of limitations instandard chromatography applications such as columns. Where one CCD orone chamber is described, it understood that multiple CCDs or chambersare also intended. Where particular components are described, it isunderstood that the individual components, pressure levels, resin orparticle types, chamber shapes, liquid, liquid flow and rotationparameters are not limiting to the invention and that novel combinationsof these and other components are included in the present invention.

As used herein, particles, beads or resins are used interchangeably andinclude any particles or materials that can be used for chromatography.A particle is capable of being used for chromatography if it functionsin a CCD to form a bed, such as in column chromatography, and acts toseparate molecules in the liquid or media that is added to the CCDchamber. The particles forming the bed are formed into a bed and heldsubstantially in one location within the chamber by the summation of thevector forces acting on the particles. Examples of particles include,but are not limited to, agarose, sepharose, silica beads, mixedcomposition beads, anionic beads, cationic beads, affinitychromatography beads and specialty beads with functional groups. Thesechromatography materials are known and are commercially available fromcompanies such as BioRad, of California, and Amersham BiosciencesPiscataway, N.J.

The present invention comprises methods and compositions used with a CCDsuch that the CCD functions as an expanded bed or packed bedchromatography device. The CCD can use adsorbents to form stablefluidized beds at high operating flow velocities. Ion exchange resinsare used, such as those made from highly biocompatible agarose basematrix with an inert crystalline quartz core material to provide therequired density. The defined particle size and density distribution ofthe adsorbents yield expanded beds with well-defined and consistenthydrodynamic properties, and with adsorption characteristics similar tothose of packed beds of standard chromatography media.

CCD methods with expanded bed characteristics can be used for initialrecovery of target proteins from crude feed-stock. The process steps ofclarification, concentration and initial purification can be combinedinto one unit operation, providing increased process economy due to adecreased number of process steps, increased yield, shorter overallprocess time, reduced labor cost and reduced running cost and capitalexpenditure. Additionally, all kinds of source materials can be used inprocessing such different materials including, but not limited to,bacterial homogenate, bacterial lysate, E. coli inclusion bodies,products secreted from bacteria, yeast, insect, animal, and plant cells,yeast cell and other cellular homogenates, whole hybridoma fermentationbroth, myeloma cell culture, whole animal cell culture broth, milk,animal tissue extracts, plant tissue extracts, unknown source materials,chemical reaction mixtures, metal slurries, and culture supernatant froma continuous fluidized bed bioreactor. Source materials are alsoreferred to as heterogenous liquids. The heterogeneous liquids can behighly heterogenous, meaning that the liquid contains very manydifferent kinds of molecules, or the heterogeneous liquid may onlycomprise more than one molecule, such as a liquid taken at a late stepin the purification process.

CCD methods and devices can provide a single pass operation in whichdesired proteins are purified from crude, particulate-containingfeed-stock without the need for separate clarification, concentrationand initial purification. The CCD bed created by the design of forceparameters allows for a distance between the adsorbent particles,providing an increased void volume fraction in the bed, which allows forunhindered passage of cells, cell debris and other particulates duringapplication of crude feed to the column.

Crude, unclarified feed, a highly heterogeneous liquid, is applied tothe CCD bed and target molecules are bound to the adsorbent while celldebris, cells, particulates and contaminants pass through unhindered.Any target molecule can be trapped this way and the method can beaccomplished using different adsorbent or absorbent materials. Weaklybound material, such as residual cells, cell debris and other type ofparticulate material, is washed out from the bed using liquid flow.Different parameters are then used to elute the captured targetmolecules from the bed using suitable buffer conditions. For example,the distance between the particles of the adsorbent material isincreased and the buffer condition is changed so that the targetmolecule is released from the adsorbent material particles and entersthe eluent phase. The eluent contains the target molecule, increased inconcentration, clarified, partly purified, and ready for furtherpurification if necessary. Thus the present invention comprises a methodfor concentrating a target molecule from a highly heterogeneous liquidcomprising adding an adsorbent material to a CCD chamber, suspending theadsorbent material by the summation of the vector forces acting on theadsorbent material, forming a chromatography bed that adsorbs the targetmolecule and allows other materials in the heterogenous liquid to passthrough the bed, eluting the adsorbed target molecule from the adsorbentmaterial and collecting the eluted target molecule. The adsorbed targetmolecule can be eluted from the adsorbent material while the adsorbentmaterial is within the chamber or chambers of the CCD or is eluted fromthe adsorbent material after the adsorbent material is outside the CCD.

CCD methods, devices and compositions comprise ion exclusionchromatography methods and materials. In ion exclusion, mobile ions withlike charge cannot penetrate the bead, which carries a fixed charge.Highly charged species are excluded from the intraparticle volume andelute sooner. In normal phase partition, the sample is distributedbetween the intraparticle (bound) water and a less polar mobile phase.By choosing an appropriate buffer, column or bed selectivity can befine-tuned for a particular compound. Nonpolar compounds are retainedmore strongly than polar compounds. In reversed phase partition, thesample molecules are distributed between a polar, usually aqueous,mobile phase and a nonpolar (aromatic) resin backbone. The morehydrophobic molecules elute later than less hydrophobic ones. Ligandexchange and size exclusion can also be used. In size exclusion,molecules too large to penetrate the effective pore structure of theresin are physically excluded from the intraparticle volume. The methodsand compositions of the present invention comprise a method forseparating molecules, comprising, forming a substantially stationary bedwithin a chamber of a CCD using compositions comprising particles andmedia, wherein the composition of particles comprises particles that aresuitable for ion exclusion and the composition of media comprise liquidsthat create an ionic environment in which a target molecule is bound bythe particles. Alternatively, the methods comprise compositions of mediain which the target molecule is not bound by the particles. In anotheralternative, the methods comprise compositions of particles having poresthat are too small for the target molecule to enter. In anotheralternative, the methods comprise compositions of particles having poresthat are sized so that the target molecule enters, and in such a method,the target molecule elutes from the CCD at a later time than if thetarget molecule had flowed directly through the bed.

Ion exchange chromatography in a CCD can comprise particles such asUNOsphere S strong cation exchange media, made by BioRad. Theseparticles are hydrophilic, spherical polymeric beads designed for theseparation of proteins, nucleic acids, viruses, plasmids and othermacromolecules. The beads are provided in 100 mM NaCl in 20% ethanol asa 50% (v/v) slurry. The beads are added to the media stream entering theCCD and the bed, made from the beads, is formed within the CCD.Determining the optimal flow rate and bed size is well within the skillof those skilled in the art and is determined by the target molecule tobe isolated or separated. The liquid containing the target molecule isadded to the CCD in the appropriate buffering conditions and flowedthrough until the target molecule is either bound to the beads orexcluded from the beads. The target molecule is then eluted directlyfrom the CCD bed if excluded, or the target molecule is eluted from theCCD bed with the appropriately buffered media.

All buffers commonly used for anion or cation exchange chromatographyare used with the ion exchange beads and methods of the presentinvention. A variety of buffers can be used in differing steps,depending on the nature of the target molecule and the heterogeneousliquid in which the target molecule is found. The use of buffering ionsthat have the same charge as the functional group on the ion exchangebeads will produce the best results. For example, phosphate ions withcation exchange beads and Tris with anion exchange beads. Cationicbuffers include, but are not limited to, acetic acid, citric acid,HEPES, lactic acid, MES, MOPS, phosphate, PIPES, pivalic acid, TES, andtricine. Anionic buffers include, but are not limited to, bicine,bis-Tris, diethanolamine, diethylamine, L-histidine, imidazole,pyridine, tricine, triethanolamine and Tris. The ion beads can beregenerated by washing with 2-4 bed volumes of 1-2 M NaCl. This washingremoves reversibly bound material.

Methods comprising reversed phase and ion-paring on silica requirecomplex eluents for effective separations. These mechanisms work on theprinciple of modifying the compound to be analyzed until it iscompatible with the bed material. Alternatively, the bed material can bemodified and the chromatographic conditions are optimized to becompatible with the compound, allowing for isocratic elution and nosample derivatization.

CCD applications contemplate the use of ion exchange methods. Ionexchange chromatography is one of the most widely used techniques forprotein purification. Two of the most commonly used ion exchangesupports are strong anion and cation exchangers. Strong anionexchangers, with quaternary amine functional groups are used forpurifying acidic and neutral proteins and peptides. Strong cationexchangers, with sulfonate functional groups, are used for purifyingbasic and neutral proteins and peptides. Any type of support materialthat can be used in the CCD device that can bear these and other ionicfunctional groups are contemplated by the present invention.

CCD Bed Materials

The type of bed material, resin, bead, all of which are interchangeableterms, that are used in the CCD methods and devices will be determinedat least in part by the chemical nature of the target molecules orcompounds and the solutions in which the target molecule is found.Certain classes of water insoluble or sparingly water soluble compoundsare preferably separated on reversed phase particle beds, while otherwater soluble compounds such as sugars, alcohols and short chain organicacids are preferably separated on the ion exchange resins. Middle rangesolubility compounds can be separated with several different methods.

A particular advantage of the CCD methods, devices and compositions isthat high resolution chromatography supports, such as smaller diameterbeads, resins or gels can be used in addition to those used inconventional columns because of the lack of gravity packing in the CCD.The density of the resin, beads or support materials can be lower thanstandard column bed materials. The input fluids or stock or fermentationbroths, which contain the target molecule in impure form can be moreviscous that is practical in standard column separations because thedensity of the bed in the CCD can be so carefully controlled andfine-tuned by rotation and other force parameters. The higher viscositycapabilities allows for less dilution of starting materials and may alsoprevent preprocessing steps such as filtration. Fluid flow is limited bythe rate of absorption, not by physical bed considerations such aminimum fluidization velocities, leading to shorter loading time.

The CCD methods, compositions and devices can be used to separate anybiomaterial or inorganic materials, that can separated bychromatographical methods. For example, CCD can be used for the analysisof proteins, carbohydrates, alcohols and organic acids in food andbeverages, biochemical, biomedical and biotechnology applications. Theparameters for separation will be different for the differing targetmolecules, the starting material and the degree of purification needed.In currently used chromatography, to achieve the high throughputrequired in industrial applications of adsorption chromatography, flowvelocities must be high throughout the complete purification cycle butwithout the beads being carried out of the column. The design andoperational parameters of the CCD permit efficient flow control withoutloss of the beads.

A method for isolating a target molecule, comprising, suspendingchromatography particles in at least one chamber in a centrifugal forcefield wherein a continuous flow of a liquid acts to create a force whichopposes the centrifugal force filed and wherein a gravitational forcecontributes to the resultant vector summation of all forces acting onthe particles, wherein the forces substantially immobilize the particlesby the summation of the vector forces acting on the particles, andforming a chromatography bed, adding a heterogeneous liquid comprisingthe target molecule, separating the heterogeneous liquid by the actionsof the chromatography bed; and retaining the separated portion of theheterogeneous liquid comprising the target molecule. This methodcomprises using any known chromatography particles, including, but notlimited to, chromatography particles that are adsorbent, size exclusion,affinity, absorbent, polar, nonpolar, cationic, anionic, ligandexchange, hydrophobic, hydrophilic, and ion-pairing, and others known tothose of skill in the art. The actions of the chromatography bed arefrom the interaction or noninteraction of the particles with thecomponents of the heterogeneous liquid and buffers that are used duringthe chromatography run of the CCD. The actions of the chromatography bedare to separate or fractionate the heterogeneous liquid in one or moreways, with the target molecule found in at least one of the fractions.

Particles

The CCD bed material can be of all types of resins or materials for theseparation methods of the present invention. Additionally, the CCD canuse novel combinations of different beads or novel combinations offunctional groups or ligands on the same bead. The particles can be, butare not limited to, very porous, macroporous, slightly porous,nonporous, hydrophilic, hydrophobic, highly charged, slightly charged,no charge, rigid, or swellable. The particles can be, but are notlimited to, plastics, methacrylate, strong anion exchangers with highbinding capacity, low binding capacity, DEAE weak anion exchangeparticles, high S strong cation exchange materials, or CM weak cationexchange materials.

A commonly used material is agarose, a material proven to work well forindustrial scale chromatography. The macroporous structure of the highlycross-linked agarose matrices combines good binding capacities for largemolecules, such as proteins, with high chemical and mechanicalstability. High mechanical stability is an important property of amatrix to be used to reduce the effects of attrition when particles aremoving freely. Because the design of the CCD allows for considerationsdifferent from column chromatography, the agarose beads may be smalleror larger or different in amount of cross-linking from standard beads.These changes or no changes are contemplated for all structuralmaterials in the bed materials. Modified agarose matrices may be lessbrittle than inorganic material such as some glass or ceramic materials.

Particles made only of organic material have limited density and wouldneed to have very large diameters for conventional chromatographyconsiderations such as high sedimentation velocity required. Such largeparticle diameters result in long diffusional path lengths, which causeconsiderable mass transfer resistance, counteracting productivity.Unlike conventional chromatography, CCD devices, methods andcompositions can employ these larger organic materials. Additionally,the present invention comprises a composite particle containing an inertcore material that is denser than organic materials. Such particles canbe designed so that their density is high at a reasonable particle size.

Particle polydispersity in bed material is also contemplated by thepresent invention. The size and density gradients position the beads atspecific locations with the CCD chamber. The smaller, lighter particlesmove to one position and the larger, heavier particles to a differentone. Polydispersity in any characteristic is contemplated by the presentinvention. Size, density, binding capabilities, exclusion pore sizes,support material differences are a few of the wide variety ofcombinations of components and factors that are used in CCD methods anddevices.

HPLC Applications

The present invention comprises methods and compositions for the CCDthat give the CCD the characteristics of high performance liquidchromatography (HPLC). The CCD methods include, among others, ionexclusion, ion exchange, ligand, exchange size exclusion, reversed phaseand normal phase partitioning, and affinity. These multiple modes ofinteraction offer a unique ability to separate compounds. The charge onthe resin provides the capability of ion exclusion, while the resinmaterial, such as polystyrene backbone, allows hydrophobic interactionto take place. The extent of the interactions depends on the compoundsbeing analyzed and the degree of selectivity required.

Reversed phase and ion pairing HPLC techniques require complex eluentconditions for effective separations. These methods work on theprinciple of modifying the compound to be analyzed until it iscompatible with the bed. Additionally, with resin-based HPLC-like CCDbeds, instead of modifying the compound to be analyzed, the bed materialis modified and rotation and other force parameters are optimized to becompatible with the compound structure. Resin-based beds allow for theuse of an isocratic HPLC system, simplifying sample preparation methodsand require no sample derivatization. This shortens sample preparationtime, and reduces total analysis time. Filtration may be the onlypreprocessing step necessary.

Affinity Chromatography

Affinity chromatography is based on the ability of the particles in thebed to specifically bind the target molecule. For example, purificationof monoclonal antibodies is one of the major applications ofchromatography and CCD methods and compositions comprise purification ofantibodies, including polyclonal and monoclonal antibodies, bindingportions, Fc regions and fragments of antibodies, and antibodyreceptors. Protein A and Protein G containing materials are means topurify various classes of immunoglobulins. For example, particles havingProtein A or Protein G are used in a CCD bed and the heterogeneousliquid comprising the target molecules, antibodies, is passed throughthe bed. The target molecule is bound by the Protein A or G and othermaterials in the heterogeneous liquid pass through the bed. Otherreceptor-based bed materials can be used to isolate species orsubclasses of antibodies.

An example of CCD methods to isolate particular monoclonals is provided.A first CCD comprising a bed material of DEAE beads with Cibacron blueF3GA dye, a mixed mode anion exchange/dye ligand, is used, which feedsdirectly into a CHT-1 ceramic hydroxyapatite chromatography bed in asecond CCD or conventional column. The DEAE-blue resin is a bifunctionalaffinity gel containing Cibacron blue F3GA dye covalently attached toDEAE agarose. The dye binds albumin and the DEAE group binds theremaining acidic proteins. This offers an alternative separation toProtein A or G binding separation. The combined dye and DEAE materialcan bind all IgG subclasses, uses mild elution conditions and providescomplete removal of all proteases. Under appropriate conditions, theantibody is eluted and the albumin is retained. The hydroxyapatite stepfurther purifies the antibody. The CHT-I bed is useful when the pI ofthe antibody is close enough to the pI of albumin to cause problems withion exchange. In addition, the of CHT-I could allow different idiotypesof the monoclonals to be separated.

Another purification method comprises use of a strong cation exchangematerial for the bed material. At a pH of 4.5 to 5, albumin isnegatively charged and does not bind. The antibody is positively chargedat this pH and binds to the bed material. The albumin is flushed out ofthe CCD chamber and the antibody is retained. A sodium chloride gradientcan be used to elute the antibody.

Another purification method comprises use of a weak anion exchanger,such as DEAE 20 weak anion exchange material. Most immunoglobulins havepIs in the 6-8 range, and a pH of 7.5 is used for the DEAE bed. Mostimmunoglobulins bind under these conditions and elute early in agradient. There is extensive literature describing weak anion exchangeconditions for antibodies, allowing for many applications of thosemethods to CCD devices and methods. With standard chromatography devicesthere is a disadvantage in using the weak ion exchanger due to therequirement for large equilibration volumes when changing pH. The CCDcan change the distance between bed particles by easily changing therotation and other force parameters and thus allows for less buffer andtime in equilibration.

Methods such as these, especially use of the blue dye affinity materialwhich can differentiate between albumin and other proteins, can be usedto separate and purify serum and plasma proteins such as complement,fetoprotein, macroglobulin, thyromedin, gelsolin and albumin. Enzymescan also be purified, including, but not limited to, kinases,dehydrogenases and other nucleotide-dependent enzymes. Enzyme substrateaffinity beads are also contemplated by the present invention.Biospecific affinity materials are used in the CCD to specificallyselect for target molecules.

Another selective binding material for affinity chromatography usesboronate-derivatized bed materials. The boronate-derivatized materialscan be made from any material used in making chromatography beads,resins or gels, such as polyacrylamide, and can be used with materialsthat are not currently used because the CCD bed materials comprisedifferent structural concerns for materials. These boronate-derivatizedbed materials are used for highly efficient separation of such lowmolecular weight compounds as nucleotides, nucleosides, catecholaminesand sugars. The boronate-derivatized bed materials have an affinity foradjacent cis hydroxyl group (cis-diols) and can separate closely relatedspecies such a AMP and cyclic AMP. Methods include separation ofcis-diol containing compounds such as cytosine, uridine and adenosinefrom one another. All of these compounds are bound but their differingaffinities permit separate elution. Size exclusion can also be combinedwith affinity, by using a bed packing density or bead exclusionparameter to separate small molecules.

Individually designed beads (bed materials) can also be used tospecifically select for certain target molecules. Bed materials thatallow for immunoglobulin coupling to an agarose or other type of supportmaterial are contemplated by the present invention. Immunoglobulins canbe attached to activated supports through primary amines or othermethods such as periodate oxidation of vicinal hydroxyls of the sugarsof the carbohydrates found on the Fc region of IgG. These specificantibodies can be directed to any target molecule and can be used forone step separation methods.

Other chemical materials can be used to purify or separate materials inCCD methods and devices. For example, chelating ion exchange resins canbe used to bind metals. An example of such a resin is a supportmaterial, such as divinylbenzene copolymers, that contain pairediminiodiacetate ions which act as chelating groups in binding polyvalentmetals such as copper, iron and other heavy metals in the presence ofmonovalent cations such as sodium and potassium. This resin has a verystrong attraction for transition metals, even in high concentrated saltsolutions. Use of such bed materials in CCD allows for environmentalclean-up methods in addition to purification of such metals or removalof such metals from biomaterials containing other target molecules.

The methods of the present invention comprise use of apparatus thatsubstantially immobilizes the particles that form the bed by use of thesummation of the vector forces acting on each particle. Embodiments ofsuch apparatus have been disclosed in U.S. Pat. Nos. 5,622,819;5,821,116; 6,133,019; and 6,214,617; and U.S. patent application Ser.Nos. 09/316,566, 09/773,027, 09/788,991, and 10/153,161, each of whichis incorporated by reference in its entirety. Though other apparatushave been used for centrifugal immobilization of particles, such as U.S.Pat. No. 4,939,087, they have been unsuccessful at long-termimmobilization of particles, cells, biocatalysts, and chromatographicmaterials because the effect of gravity is ignored. Thoughmicro-organisms or animal cells are quite light in weight, their mass isnon-zero. Consequently, gravity has a significant effect on theparticle, and this effect will increase with time. Over longer timeperiods, the weight of the suspended particles causes these particles tosettle to the lowest regions of the biocatalyst immobilization chamber,disrupting the balance of forces which initially suspended them in thechamber. Further, the aggregation of these particles into a largerparticle with virtually the same density as the individual particlesresults in an increased centrifugal effect which causes the aggregatesto migrate to longer radii, eventually clogging the liquid input port.

The apparatus used in the methods of the present invention takeadvantage of the relationships inherent in (1) Stoke's Law and thetheory of counterflow centrifugation; (2) the geometrical relationshipsof flow velocity and centrifugal field strength; (3) Henry's Law ofGases; and, (4) the effect of hydraulic pressure on media and particles.The methods of the present invention comprise apparatus that are capableof forming chromatography beds by the immobilization ofthree-dimensional arrays of particles, such as known chromatographicbeads and resins.

The theoretical basis of the process utilized by the apparatus of thepresent invention utilizes a novel method to immobilize particle arrays.A proper application of Stoke's Law in combination with provision forthe effect of gravity which also acts on the immobilized particlesresults in a mathematical relationship which allows for the relativeimmobilization of such particles. The effect of gravity can becompensated for by an alternative choice of rotational axis as is shownin FIG. 1. If rotation about the horizontal axis (y) is chosen insteadof rotation about the vertical axis (z), as is most common in biologicalcentrifugations, then the effect of gravity on immobilized particleswill always be limited to action solely in the x-z plane. Since this isthe same plane in which both the centrifugal as well as the liquid flowrelated forces are constrained to act, the motion of a restrainedparticle at any point in a rotational cycle is the resultant of the sumof the three types of forces acting upon it.

As is shown in Inset A of FIG. 2, where the plane of the Figure is thex-z plane, the effect of gravity (Fg) on the position of a particlesuspended in a radially-directed centrifugal field (Fc) while an exactlyequal and opposing force supplied by an inwardly-directed flowing liquid(Fb) is directed toward the particle, can be calculated by theevaluation of equations 1-4 where (k) represents the downwarddisplacement in the x-z plane imparted by gravitational forces during anangular rotation of the rotor position equal to (a). Analysis of themotion of a particle under these constraints and for [2□×(k/a)]<R (a lowmass particle) results in the determination that the motion is periodic;that is, the particle motion results in a return to its starting placeafter a complete rotation of 360 degrees (after equilibrium is reached).As is shown in FIG. 2, the effect of gravity on the motion of a particleotherwise immobile as a result of the opposing equality of thecentrifugal and flow-related forces results in a decrease in radialposition in quadrants I and II, and an exactly equal radial lengtheningin quadrants III and IV. Thus, the radial distance of the particle fromthe axis of rotation also exhibits a periodic motion over the course ofa full rotation of 360 degrees. It should be noted that, mathematically,measurement of the periodicity of motion requires only one rotation ifmeasurement begins at either 90 or 180 degrees whereas two fullrotations are required if measurement begins at either zero or 180degrees, since a new equilibrium radial distance different from theoriginal results in the latter case.

The effective motion of a particle through a complete rotational cycleis shown in the inset of FIG. 3. If the sides of a container in whichthe particle is suspended are labeled 1 and 2, then the motion of theparticle over the course of one rotational cycle would describe a circlewith its center displaced toward the “leading edge” side of theparticle's container. Thus, a particle suspended in a centrifugal fieldwhich is opposed by an equal liquid flow field will be constrained toperiodic motion (and thus is effectively immobilized) if the balance ofthe radially-directed forces can be maintained over the course of itsmovement.

A graphical representation is shown in FIG. 4, in which the axis ofrotation is now the (y) axis. Under these conditions the hypothesis ofSanderson and Bird can now be restated and applied to long-termimmobilization of particles. There is a radial distance along the z axis(rz) which, when evaluated by Eqn. 3, represents a position in which theparticle is relatively immobilized in a centrifugal field which isexactly opposed by an inwardly-directed liquid flow, even in thepresence of a gravitational field. Furthermore, a simplification ofStoke's Law (Eqn. 1) under the conditions of uniform particle size,shape, and density and a homogeneous liquid flow results in Eqn. 2,where it is obvious that the Sedimentation Velocity of a particle (SV)is a simple linear function of the applied centrifugal field. Similarly,Eqn. 3 can then be rewritten under the same conditions to yield Eqn. 4,where liquid Velocity (V in Eqn. 3) has been replaced by liquid FlowVelocity (Fv). Equation 4 suggests that there is a continuum of liquidflow velocities and applied centrifugal fields which could be matched bythe evaluation of constant (C), all of which would satisfy therequirement of relative particle immobilization. Further, if the liquidflow velocity could be varied as a function of (z), there could be aseparate application of this equation at each radial distance.Consideration of the implications of Eqn. 4 is important for therelative immobilization of three-dimensional arrays of particles asopposed to the immobilization of two-dimensional arrays of particles ata single radial distance from the rotational axis.

If the chamber in which a particle is located is cylindrical (as isgraphically depicted in FIG. 5) and if a liquid is flowed into thischamber from the end of the chamber most distal to the axis of rotation,then it is obvious that the flow velocity of this liquid flow (asdefined in Eqn. 1, FIG. 5) will have a single value at all points notoccupied by layers of particles. As a consequence, if a two-dimensionalarray of particles is in positional equilibrium at a particular radialdistance (A1), as is indicated in Eqn. 2, (where CF is the centrifugalfield strength and FV is the liquid flow velocity) then particles forcedto occupy positions at radial distances either greater than or smallerthan A1, such as those located in FIG. 5 at A2 or A3, will necessarilybe presented with an inequality of restraining forces which will resultin net translation of the particles. Thus, those particles located atA2, a longer radial distance than A1, will experience a greatercentrifugal force than those at A1 and will necessarily migrate tolonger radial distances (Eqn. 3). Conversely, particles initiallylocated at A3 would experience a reduced centrifugal field and wouldmigrate to shorter radial distances (Eqn. 4). Thus, it is not possibleto form a three-dimensional array of particles in a parallel-walledchamber such as that of FIG. 5.

If, however, the biocatalyst immobilization chamber has a geometry suchthat its cross-sectional area increases as the rotational radiusdecreases, as is graphically displayed in FIG. 6, then it ismathematically possible to form three-dimensional arrays of immobilizedparticles. This is a consequence of the fact that the microscopic flowvelocity of the liquid flow varies inversely as the cross-sectional area(Eqn. 1) while the relative centrifugal field varies directly as therotational radius (Eqn. 2). Thus, if values of flow velocity androtation velocity are chosen such that a two-dimensional array ofparticles is immobilized at rotational radius A1 (Eqn. 3), then it ismathematically possible to adjust the “aspect ratio” of the side wallsof the biocatalyst immobilization chamber such that those particlesinitially located at radial distance A2 could also experience either ansimilar equality of forces or, as is shown in Eqn. 4, an inequality offorces which results in net motion back toward the center of thechamber. A similar argument may be applied to particles located at A3(see Eqn. 5). Although the geometry of the biocatalyst immobilizationchamber as depicted in FIG. 6 is that of a truncated cone, note thatother geometries could be alternatively used—subject to the constraintthat the cross-sectional area of the chamber increases as the rotationalradius decreases. Thus, as is depicted in FIG. 7, it is possible toconstruct a three-dimensional array of particles in a varyingcentrifugal field opposed by a liquid flow field if the biocatalystimmobilization chamber geometry chosen allows for a flow velocitydecrease greater than or equal to the centrifugal field strengthdecrease as the rotational radius decreases. In the geometry chosen inFIG. 7, that of a truncated cone, the two-dimensional arrays ofparticles at each rotational radius (Rc) will each be constrained tomotion toward that radius where the opposing forces are exactly equal.

While, at first glance, the description presented above would suggestthat the net effect of the mismatch of forces at all radii other thanthat which provides immobilization would result in a “cramming” of allparticles into a narrow zone centered on the appropriate radius, such isnot the case. As is shown graphically in FIG. 8, as each layer ofparticles approaches an adjacent layer, it will move into a region wherea “cushioning effect” will keep each layer apart (the horizontal arrowsin FIG. 8). The explanation for the inability of adjacent layers ofparticles to interdigitate is a consequence of an analysis of themicroscopic flow velocity profile through each layer. In FIG. 9, asingle representative stratum of spherical particles confined to aparticular radial distance in a chamber layer of circular cross-sectionis presented. The ratio of the diameters of the particles to thediameter of the cross-section of FIG. 9 is 12:1. While the magnitude ofthe flow velocity of the liquid through unoccupied portions of thechamber cross-section can be quantified simply from the chamberdimensions at that point, the flow velocity through a region occupied bya stratum of particles will necessarily be much greater than that in theabsence of a stratum of particles because of the greatly reducedcross-sectional area through which the liquid must travel. As is shownin the graph in FIG. 9, the increase in flow velocity through a stratumof the above dimensions is more than double that determined in the freespace just adjacent to the stratum on each side. This microscopicincrease in local flow velocity in the region of each stratumeffectively provides a “cushion” which keeps each adjacent stratumseparate.

In actual use, it has been determined that, for the case of a chambergeometry of a truncated cone, it is preferable that the most distalregion of the truncated cone be the region where an exact equality ofcentrifugal forces and liquid flow velocity is achieved. The “aspectratio” (the ratio of the small radius of the truncated cone to the largeradius of the truncated cone) of the truncated cone is determined by thesimultaneous solution of the two equations presented in FIG. 10. In Eqn.2, the desired boundary condition of immobility for that “lowest”stratum of particles is presented. It states that the intrinsicsedimentation rate of the particle due to gravity (SR) times therelative centrifugal field applied at that radial distance (RCF) beexactly equal to the magnitude of the liquid flow velocity (FV) at thatpoint. In Eqn. 1, a desired boundary condition at the opposite surfaceof the array of particles is presented. In order to insure retention ofall particles within the biocatalyst immobilization chamber, a boundarycondition wherein the product of SR and RCF is twice the magnitude ofthe flow velocity at that radial distance has been arbitrarily chosen.Simultaneous solution of the desired boundary condition equations isused to solve for the ratio of the conic section diameters when theupper diameter and conic length is known.

FIG. 11 is a profile of the relative magnitudes of the flow-relatedforces and the centrifugal forces across a biocatalyst immobilizationchamber of conical cross-section which has dimensions in this example oflarge diameter=6.0 cm, small diameter=3.67 cm, and depth=3.0 cm. Wedefine the Relative Sedimentation Rate as the product of the intrinsicsedimentation rate of a particle due to gravity in a nutrient media atits optimal temperature and the applied centrifugal field. For a givenflow rate (in this example 10 mL/min) into a biocatalyst immobilizationchamber of the indicated dimensions, where the proximal end of thebiocatalyst immobilization chamber is 9.0 cm from the rotational axis,the product of the intrinsic particle sedimentation rate due to gravityand the angular velocity is a constant at the given flow rate in orderto satisfy the desired boundary conditions (see FIG. 10). In otherwords, the angular velocity need not be specified here since its valuedepends only on the particular particle type to be immobilized. Thedotted line in FIG. 11 displays the linear variation in the centrifugalfield strength from the bottom to the top of the biocatalystimmobilization chamber, while the solid line displays the correspondingvalue of the flow velocity. At the bottom of the chamber (the mostdistal portion of the chamber), the forces are equal and a particle atthis position would experience no net force. At the top of the chamber,a particle would experience a flow-related force which is only one-halfof the magnitude of the centrifugal field and would thus be unlikely toexit the chamber, even in the presence of a nearby region of decreasingcross-sectional area (the chamber liquid exit port), where flowvelocities will increase markedly.

It should be clear from the foregoing that, subject to the necessarycondition that the cross-sectional area increases as rotational radiusdecreases, there are other geometrical chamber configurations whoseshape could be manipulated in order to establish boundary andintermediate relationships between the applied centrifugal field and theliquid flow velocity forces at any radial distance in order to establishdesired resultant force relationships in the three-dimensional particlearrays. In practice, however, it is undesirable to utilize geometrieswith rectangular cross-sections as a result of the anomalous effects ofcoriolis forces which act in a plane transverse to the rotational plane.In the case of rectangular cross-sections, these otherwise unimportantforces can contribute to interlayer particle motion.

It should also be clear from the foregoing that the effect ofgravitational forces acting on the individual particle masses which actsindependently of the applied centrifugal forces are even less importantthan was indicated earlier. In particular, since the basic effect ofgravity on an otherwise immobilized particle is to either cause radiallengthening or radial shortening, such a motion of a particle willnecessarily bring it either into a region of increased flow velocitymagnitude (longer radii) or decreased flow velocity magnitude (shorterradii) with only a much smaller change in centrifugal field strength.

As a consequence, the periodic motion of a particle due to gravitationaleffects on its intrinsic mass will be severely dampened in the presenceof such unbalanced opposing force fields and will amount to, in the caseof low mass particles, a vibration in place.

It is preferred to control either the introduction of, or the generationof, gases within the immobilization chamber. One may ensure thiscondition by the application of Henry's Law, which, in essence, statesthat the quantity of a gas which may be dissolved in a liquid is afunction of the system pressure. Thus, if the hydraulic pressure of theliquid-containing parts of the system, including chamber and the liquidlines leading to and from the chamber, are maintained at a hydraulicpressure sufficient to fully dissolve the necessary quantity of inputgas and to insure the solubility of any produced gases, then there willbe no disturbance of the immobilization dynamics.

FIG. 12 illustrates a system according to various embodiments of theinvention. In a system 100 utilizing methods and compositions forseparating and isolating target molecules, such as in chromatography, aCCD 102 operates in conjunction with a production vessel 104 and a meansfor product capture 106.

Typically, a CCD 102 operates independent of an immobilized particlesize or particle function. That is, a CCD 102 can be operated atparticular liquid flow rate and revolutions per minute (RPM)combinations where arrays of, for example, either 5 or 200 μm diameterion exchange resin beads or gel exclusion beads are immobilized; and (2)the backpressure or the resistance to liquid flow through an array ofimmobilized particles is a small fraction of the backpressure of anequivalent packed bed of the same number of particles.

A production vessel 104 can be a conventional device associated withknown methods and systems for producing, storing, or otherwise providinga starting material, or heterogeneous liquid, to a chromatographicdevice such as a CCD 102.

A means for product capture 106 can be a conventional device associatedwith known methods and systems for capturing, storing, or otherwisereceiving one or more products from a chromatographic device such as aCCD 102.

As shown in FIG. 12, a CCD 102 receives the starting material,heterogeneous liquid, produced in a production vessel 104. The CCD 102captures or otherwise isolates target molecules from the startingmaterial using an appropriate or suitable chromatographic technique. TheCCD 102 then provides the target molecules to the means for productcapture 106. As shown in 108 of FIG. 12, depending upon thechromatographic technique used by the CCD 102, the CCD 102 isolatestarget molecules from other materials in the heterogeneous liquid, shownin the inset chromatogram, where peaks indicate the separation ofdifferent materials in the heterogeneous liquid.

FIG. 13 illustrates another system 200 utilizing methods andcompositions for separating and isolating target molecules, such as inchromatography, in accordance with various embodiments of the invention.In this embodiment, one or more CCDs 202 a,b can be used in respectivechromatographic processes to obtain desired product molecules. Each CCD202 a,b includes an immobilized chromatographic particle array,chromatographic bed, in accordance with a chromatographic process. Thefirst CCD 202 a can be used as a primary process device, and the secondCCD 202 b can be used as a backup or overflow process device. Startingmaterial, heterogeneous liquid, from a common production vessel 204 canbe pumped to a first CCD 202 a and then to a second CCD 202 b. Aneluting medium 206 such as a liquid of a chemical composition designedto cause the elution of the target molecules from an immobilizedchromatographic particle array, can be provided to each CCD 202 a,b asneeded. Typically, when the fluid flow from the first CCD 202 a reachesa particular capacity, subsequent fluid flow is diverted to the secondCCD 202 b. Respective product capture reservoirs 208 a,b connect to eachCCD 202 a,b to collect target molecules from the chromatographicprocesses implemented by the CCDs 202 a,b. A non-product waste 210, suchas a liquid, is output from each CCD 202 a,b as a result of thechromatographic processes. Typically, a collection device or storagereservoir captures the non-product waste 210.

For example, the continuous flow-type system 200 can be used inconjunction with classical ion exchange-type or affinity-typechromatography processes. Using these types of processes in conjunctionwith the CCDs shown in the continuous-flow system 200, target moleculescan be obtained from a heterogeneous liquid.

A continuous flow-type process implemented by the system 200 isdescribed below. Initially, a starting material, heterogenous liquid,containing target molecules is introduced such as being pumped from aproduction vessel 204, to a first CCD 202 a. The target molecules areinitially adsorbed by the first CCD 202 a in a chromatographic particlearray made from resin particles. The target molecules become immobilizedwithin the first CCD 202 a. When all available binding sites aresaturated, an eluting medium 206 subsequently elutes the desired productmolecules from the support. Waste liquid from the first CCD 202 a isthen diverted to a waste reservoir or to non-product waste 210. In thismanner, a more purified and concentrated product may be continuouslyextracted from the production vessel 204.

When the first CCD 202 a is about to reach a predefined target moleculebinding capacity, the flow from the production vessel 204 is diverted toflow through a second CCD 202 b. Output of target molecules from thefirst CCD 202 a can then be diverted to product capture 208 a where atarget molecule is collected, as shown by the peak in a chromatogram.Output of target molecules from the second CCD 202 b can be diverted toa second product capture means 208 b. Thus, when the target moleculebinding capacity reaches a predefined amount for either the first CCD202 a or second CCD 202 b, flow can be diverted to the other CCD asneeded. In this manner, the first. CCD 202 a and the second CCD 202 bcan be operated in alternating periods to provide a continuous flow ofdesired product molecules.

FIG. 14 illustrates another system 300 utilizing methods andcompositions for separating and isolating target molecules, usingchromatography, in accordance with various embodiments of the invention.In this embodiment, one or more CCDs are used in a continuous processflow-type scheme in which high molecular weight product molecules can beisolated and purified from a heterogeneous liquid. Similar to the system200 in FIG. 13, the system 300 provides a heterogeneous liquid from aproduction vessel 302 to a first CCD 304. In the first CCD 304, thecellular portion of the heterogeneous liquid is removed. Waste isdiverted to a reservoir such as non-product waste 306. Next, thecell-free liquid is passed from the first CCD 304 to a second CCD 308,where low molecular weight protein contaminants are discarded and theliquid containing target molecules is passed to downstream purificationsections. Waste from the second CCD 308 is diverted to another reservoiror to non-product waste 306. Next, a third CCD 310 and fourth CCD 312are operated alternatively to first absorb and then elute a morepurified and concentrated protein target molecule. Eluent 314 may beadded to each of the third CCD 310 and/or fourth CCD 312 as needed.Waste from each of the third CCD 310 and fourth CCD 312 is diverted toseparate or common reservoirs such non-product waste 316. A fifth CCD318 and sixth CCD 320 are employed in alternative operation to adsorband elute the protein product from affinity chromatography resin arrays.Eluent 322 may be added to each of the fifth CCD 318 and/or sixth CCD320 as needed. Waste from each of the fifth CCD 318 and sixth CCD 320 isdiverted to separate or common reservoirs such non-product waste 316.The resultant product stream is an output 324 in which the proteintarget molecule has undergone at least four sequential chromatographicpurification steps.

FIGS. 15-24 illustrate views of a device or apparatus according to anembodiment of the invention, also generally known as a CentrifugalChromatography Device or “CCD.” The embodiment shown is directed to anapparatus for substantially separating and isolating target molecules,such as in chromatography. Depending upon the type of target molecule tobe separated and isolated, and the role of the CCD in a particularchromatographic process, various chromatography resin arrays can beutilized with a CCD.

FIG. 15 illustrates a front view of a CCD 400 with at least one chamber402 for separating and isolating at least one biomaterial. In thisembodiment, the CCD 400 includes at least one chamber 402 positionedalong a longitudinal axis of a shaft 404. Note that the CCD 400 mayinclude any number of chambers 402 mounted to the shaft 404. Turning toFIGS. 21A and 22A, the shaft 404 typically has an input cavity 406, anoutput cavity 408, an injection orifice 410, and an output orifice 412.The shaft 404 is typically composed of a stainless steel, typically 304or 316 stainless steel annealed, ground and polished. However, the shaft404 may be composed of metals including, but not limited to, steel,iron, and titanium, plastics, composites, combinations thereof, or anymaterial capable of withstanding stresses developed in the CCD 400during operation.

The input cavity 406 and the output cavity 408 preferably are positionedwithin the shaft 404 and extend throughout the length of the shaft 404.The injection orifice 410 and the output orifice 412 are in fluidcommunication with the input cavity 408 and the output cavity 410,respectively, and each orifice contacts an exterior surface of the shaft406. The CCD 400 further includes at least one injection element 414which is in fluid communication with the injection orifice 410 andpositioned within each chamber 402, as shown in FIGS. 15 and 16A-B. Inthis embodiment, a plurality of injection elements 414 are shown in FIG.16A. Additionally, the CCD 400 includes a means for rotating the shaft404, such as a motor (not shown), and the at least one chamber 402 aboutthe longitudinal axis of the shaft 404.

As shown in FIGS. 15-20, a chamber 402 typically includes two sides 416,which may be composed of a material such as stainless steel.Alternatively, each side 416 may be composed of any material capable ofwithstanding the stresses developed during operation of the CCD 400, andmay include, but is not limited to metals such as iron or titanium,plastics, composites and/or combinations thereof. Note that in thisembodiment, when the sides 416 of the chamber 402 are fit together thateach chamber 402 has an internal cavity 418 in the shape of a triangulartoroid when viewed from the side. Furthermore, the external shape of thechamber 402 is desirably round and wheel-shaped when the two sides 416are fit together. The outermost portion of the internal cavity 418maintains an angled portion 420 between each interior surface of thechamber 402 when viewed from a position generally orthogonal to thelongitudinal axis of the shaft 404. The angled portion 420 may typicallyhave an angle of about 0 to 90 degrees, and is preferably about 25degrees.

The angled portion 420 should be such that when the CCD 400 is inoperation, a biomaterial (not shown) that is contained within thechamber 402 forms a substantially stationary chromatographic materialwhich does not contact the exterior surface of a manifold sleeve 422 orthe shaft 404. Further, the chamber 402 can include a transition sectionor walls between the angled portion of the chamber and the sleeve orshaft. Typically, the transition section is composed of a surface thatis generally orthogonal to the longitudinal axis of the shaft 404.Positioning the transition section in this fashion discourages thebiomaterial from contacting the manifold sleeve 422 during operation ofthe CCD 400 thereby allowing the biomaterial to perform its intendedfunction.

The sides 416 of the chamber 402 are typically fastened together using aplurality of bolts 424. The bolts 424 are positioned within holes 426located around the perimeter 428 of the chamber 402. Alternatively, eachside 416 of the chamber 402 may be held together using any assortment offasteners or other releasable connection mechanisms. Once the sides 416of the chamber 402 have been assembled together, the width of theinternal cavity 418 of the chamber 402 may be approximately 2.6 inches(6.7 cm) with the diameter of the chamber 402 being approximately 12.0inches (30.5 cm), and the diameter as measured between opposing bolts424 is approximately 9.8 inches (25.0 cm). However, other embodiments ofthe CCD 400 may include a chamber 402 having dimensions in accordancewith the scope of this invention, as set forth above.

A seal between each side 416 may be established using an o-ring 430,which is typically positioned on the interior surface of a recessedportion 432 of the internal cavity 418 of the chamber 402.

Once the sides 416 are assembled, the o-ring 430 contacts both sides416. Alternatively, the seal between each side 416 of a chamber 402 maybe created using means including, but not limited to, a releasableadhesive, a gasket or any type of sealant material.

As shown in FIGS. 15, 21A-B, 22A-B, the shaft 404 can be divided intotwo portions that each mount to the chamber 402 at or near the centralportion of a respective side 416 of the chamber 402. A flange 434 oneach portion of the shaft 404 permits the shaft 404 to connect to theexterior surface of the chamber 402. Bolts 436 are positioned withinholes 438 located in the flange 434 and machined into the exteriorsurface of the chamber 402. Alternatively, the shaft 404 may be securedproximate to the chamber 402 using any assortment of fasteners or otherreleasable connection mechanisms. Once the shaft 404 has been connectedto the sides 416 of the chamber 402, the shaft 404 can then be driven torotate the shaft 404 which transmits its rotational force through theflange 434 and to the chamber 402.

Referring now to FIGS. 23A-E, a manifold sleeve for a chamber is shown.A chamber 402 may include a manifold sleeve 422 having an input channel440, at least one output channel 442 in an inner wall of the manifoldsleeve 422, a plurality of input apertures 444, and a plurality ofoutput apertures 446 extending between the input channels 440 and outputchannels 442 respectively and an outer wall of the manifold sleeve 422.Preferably, the input channel 440 is positioned at a midpoint of alongitudinal axis of the manifold sleeve 422. Alternatively, the inputchannel 440 may be positioned at any point along the longitudinal axisof the manifold sleeve 422. In the preferred embodiment, the inputchannel 440 is positioned between a plurality of output channels 442.O-rings 448, shown in FIGS. 15 and 16B, are typically located at arecessed edge 450 along the outer wall of the manifold sleeve 422.Furthermore, O-rings 448 may be positioned adjacent to the shaft 402 andaround the injection orifice 410 and the output orifice 412. The o-rings448 provide a seal, to prohibit fluid flow between the shaft 404 and thechamber 402.

The manifold sleeve 422 is positioned in the chamber 402 so that theinput channel 440 of the manifold sleeve 422 is in fluid communicationwith the injection orifice 410 of the shaft 404, and each output channel442 of the manifold sleeve 422 is in fluid communication with eachoutput orifice 412 located within the shaft 404. In such a position, theo-rings 448 located between the manifold sleeve 422 and the sides 416 ofthe chamber 402 form a seal which prevents the input fluid from mixingwith and contaminating the output fluid. The plurality of inputapertures 444 extend from the input channel 440 to the outer wall of themanifold sleeve 422. Similarly, the plurality of output apertures 446extend from the output channel 442 to the outer wall of the manifoldsleeve 422.

The manifold sleeve 422 is sized to fit completely within the chamber402, once both sides 416 of the chamber 402 have been fastened together.Of course, there are numerous ways to position the manifold sleeve 422relative or proximate to the shaft 404 as would be understood by one ofordinary skill in the art. Furthermore, it will be understood by one ofordinary skill in the art that there is more than one way to accomplishplacing the CCD 400 in fluid communication with a source of input fluid.

From the internal cavity 418 of the chamber 402, as shown in FIGS. 15,16A-B, and 19B, fluid can exit the chamber 402 via a plurality ofchamber output apertures 452. From these apertures 452, fluid travelsthrough a chamber output channel 454 to the output aperture 412 and thenthrough the shaft 404 via the outlet cavity 408, where the fluid can becollected from the CCD 400.

As shown in FIG. 15, the CCD 400 is connected to the shaft 404 with adrive pulley 456. In FIG. 24, the CCD 400 mounts to a stand 458 and amotor 460 is connected via a pulley belt (not shown) to drive the drivepulley 456. The drive pulley 456 is mechanically fastened to the shaft404, preferably using a weld, an adhesive, a keyway, or othermechanical-type connection. The stand 458 positions the shaft 404perpendicular to a gravitational force which is typically accomplishedby locating the shaft 404 parallel to the Earth's surface. The stand 458is designed to restrict the shaft 404 from any movement, exceptrotational movement, about the longitudinal axis of the shaft 404. Thestand 458 mounts to bearing assemblies 462 which allow the shaft 404 torotate while maintaining its position. In operation, the motor 460 isused to rotate the shaft 404 and one or more chambers 402 attachedthereto about the longitudinal axis of the shaft 404. The motor 460 iscapable of rotating the shaft 404 at any rate desired by the user.

In operation, the chamber 402 houses a chromatographic material,positioned between the exterior surface of the manifold sleeve 422 andthe interior surface of the chamber 402. The motor 460, together withthe pulley belt and drive pulley 456, rotate the shaft 404 and at leastone chamber 402 at a desired rate. As shown in FIG. 24, the CCD 400typically includes a shield or safety containment chamber 464 which mayinclude two halves and may be hinged or bolted at opposing ends of thestand 458 in order to allow for easy removal of the shield or safetycontainment chamber 464. The shield or safety containment chamber 464provides a thermal barrier or heat containment device for maintaining aconstant temperature inside the shield or safety containment chamberwhere the chamber 402 contains the chromatographic material.Furthermore, the shield or safety containment chamber 464 protectsindividuals from contacting, the rotating chambers 402. As the chamber402 is rotated, pressurized fluid is typically delivered to each chamber402 via an input feed tube 460, the input cavity 406, the injectionorifice 410, the plurality of input apertures 444, the plurality ofoutput apertures 446 and the plurality of injection elements 414. Thepressure of the fluid may be monitored using a pressure gauge. Theinjection elements 414 release the pressurized fluid proximate to theinterior surface of the internal cavity 418 of the chamber 402preferably located the furthest distance from the longitudinal axis ofthe shaft 404.

After the fluid has been released, the fluid flows from the outermostportion of the internal cavity 418 of the chamber 402 inwardly towardthe plurality of chamber output apertures 452 located on the interiorcavity 418 of the chamber 402 adjacent to the manifold sleeve 422. Whenthe design of the chamber 402 is a triangular toroid, as set forthabove, the fluid injected into the chamber 402 decreases in velocity asit moves from the outermost portion of the internal cavity 418 of thechamber 402 inwardly toward the longitudinal axis of the shaft 404. Thevelocity of the fluid is reduced because the cross-sectional area of thechamber 402 increases in size moving from the outermost portion of theinternal cavity 418 of the chamber 402 toward the longitudinal axis ofthe shaft 404. Injecting the pressurized fluid at the outermost portionof the internal cavity 418 of the chamber 402 positions the fluid sothat it must diffuse through the biomaterial before it leaves thechamber 402 via the plurality of chamber output apertures 452. Fromthese apertures 452, the fluid travels through a chamber output channel454 to the output aperture 412 and then through the shaft 404 via theoutlet cavity 408, where the fluid can be collected from the CCD 400.

The CCD 400 positions and suspends the particles that form achromatography bed for various beneficial purposes. The CCD 400 mayinclude a particles composed of one or more components capable ofperforming chromatographical functions or processes.

As mentioned above, the CCD 400 may include a plurality of chambers 402located adjacent one another on a single shaft 404.

However, the chamber 402 or plurality of chambers 402 may be increasedin diameter, while maintaining their triangular toroidal shape, toimmobilize and maintain other biomaterials or materials used forchromatographical functions or processes.

Thus, the present invention comprises methods of separation of targetmolecules comprising compositions comprising chromatography beads,particles, resins or gels that are used in apparatus described herein.Many embodiments are disclosed herein, and combinations of methods,compositions and apparatus are contemplated by the present invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “compound” is a referenceto one or more such compounds and includes equivalents thereof known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed above and throughout the text areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention.

It is to be understood that this invention is not limited to theparticular methodology, protocols, particles, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

EXAMPLE 1 Formation of a Chromatography Bed

An analytical-scale CCD unit equipped with a Model 101096 transparentacrylic immobilization chamber (total volume=30 mL) was loaded with 2 mLof 30 μm diameter glass beads (Cat. No. GP0029, Whitehouse Scientific,UK. The CCD unit was turned on and a RPM of 350 and a flow of 50%-50%(v/v) glycerol—0.01 M sodium phosphate buffer (ph=7.0) was initiated.The physical appearance of the immobilization chamber and its contentscould be continuously observed by means of stroboscopic illumination.

At flow rates below 5 mL/min, the glass beads formed a classical packedbed at the long-radius terminal portion of the chamber. As the flow ratewas increased to 7 mL/min, the packed bed expanded to an estimatedvolume of 3 mL and became brighter by visual examination. Stepwiseincreases in liquid flow rate from 7-12 ml/min resulted in virtuallyimmediate stepwise expansions of the bed volume and an increase in bedbrightness. Similarly, stepwise flow rate reductions resulted in bedcontraction and appearing to darken. At all flow rates between 7 and 12mL/min, there was a clearly observable division between the short-radiusterminus of the array of glass beads and the flowing liquid exiting thechamber at its short-radius output port.

In order to assess the homogeneity of the bed formed by the methodoutlined above, the liquid medium was changed to 50%-50% (v/v)glycerol—0.01 M sodium acetate buffer (ph=5.0) and a 2 mL packed bed ofglass beads was expanded to an apparent volume of 4 mL, demonstratingthat bed fluidization was not dependent on either buffer chemical orliquid pH. Next, a 100 ml quantity of buffer containing 1% Trypan bluewas prepared and flowed into the CCD at 7 ml/min. After a short delay,the entrance of the blue-colored liquid medium into the immobilizationchamber was observed. The progress of the blue dye front as it migratedanti-radially through the expanded bed could be observed. The progressof the dye front was very regular with no evidence of channeling orother flow irregularities in the fluidized bed.

The ability to expand and contract the glass bead array through multiplecycles by means of flow rate changes at constant RPM and the regularityof the dye penetration of the bed were taken as strong evidence that aclassical fluidized bed had been formed.

EXAMPLE 2 Ion Exchange Chromatography

The ability of a bed of 30 μm glass beads immobilized in ananalytical-scale CCD unit to exhibit ion-exchange chromatographicproperties was assessed in, the following manner. The CCD was operatedat RPM=350 and a 7 mL/min flow of 50%-50% (v/v) glycerol—0.01 M sodiumphosphate buffer (ph=7.0) was initiated after 2 mL of glass beads hadbeen placed in the chamber. After bed formation had been demonstrated(flow rate increased to 10 mL/min; saw subsequent bed volume rise;decreased flow rate to 7 mL/min; saw bed volume lower) a 100 mL quantityof 1% trypan blue in 50%-50% (v/v) glycerol—0.01 M sodium phosphatebuffer (ph=7.0) was pumped into the immobilization chamber. As thissolution passed through the immobilization chamber, the clear, colorlessbeads took on a dark blue color. After about 15 min the liquid flow intothe CCD was replaced with 50%-50% (v/v) glycerol—0.01 M sodium phosphatebuffer (ph=7.0) without the dye material. The glass bead array waswashed clear of unbound dye for an additional 15 min. While the liquidflow into and out of the CCD unit was now clear and colorless, thefluidized bed of glass beads were still darkly stained with bound dyemolecules, slightly less darkly than they were prior to the 15 min.wash. Next, the input liquid flow into the CCD was replaced with 50%-50%(v/v) glycerol—0.1 M sodium acetate buffer (ph=5.0). As this solutionentered the immobilization chamber, the blue stain on the glass beadsbegan to fade. After 100 mL had flowed through the chamber, theimmobilized glass beads were again clear and colorless to the naked eye.These results suggest that the cationic blue dye binds with someaffinity to the anionic glass surface at neutral (and likely also atbasic) pHs. As the pH of the flowing liquid is lowered, these data showthat the increased hydrogen ion concentration in the flowing liquidresults in release of the bound dye into the liquid flow.

Those skilled in the art will now see that certain modifications can bemade to the invention herein disclosed with respect to the illustratedembodiments, without departing from the spirit of the instant invention.And while the invention has been described above with respect to thepreferred embodiments, it will be understood that the invention isadapted to numerous rearrangements, modifications, and alterations, allsuch arrangements, modifications, and alterations are intended to bewithin the scope of the appended claims.

What is claimed is:
 1. An apparatus for isolating a target molecule,comprising: at least one rotating chamber with an inlet port and anoutlet port, the chamber containing a plurality of chromatographyparticles in an interior thereof and rotating about a horizontal axis tocreate a centrifugal force on the particles; a first pressurized liquidsource containing a first liquid, the first pressurized liquid source influid communication with the inlet port and configured to flow the firstliquid through the inlet port, through the chamber, and out of theoutlet port, such that a liquid flow force is imparted on thechromatography particles inside the chamber, wherein the liquid flowforce substantially opposes the centrifugal force, wherein agravitational force contributes to a resultant vector summation of allforces acting on the chromatography particles, and wherein thegravitational, liquid flow, and centrifugal forces substantiallyimmobilize the chromatography particles in a fluidized chromatographybed inside the chamber; and a second pressurized liquid sourcecontaining a heterogeneous liquid comprising the target molecule, thesecond pressurized liquid source in fluid communication with the inletport and configured to flow the heterogeneous liquid through the inletport and to the fluidized chromatography bed; wherein the outlet port isconfigured to discharge the heterogeneous liquid less the targetmolecule.
 2. The apparatus of claim 1, wherein the chamber is configuredto: adjustably rotate to affect either the density or shape of thefluidized chromatography bed inside the chamber.
 3. The apparatus ofclaim 1, wherein at least one chamber comprises a plurality of chambers.4. The apparatus of claim 1, wherein the chamber comprises a truncatedcone portion.
 5. The apparatus of claim 1, wherein the chromatographicparticles comprise an adsorbent material, a resin, an agarose particle,a silica particle, an ion exchange particle, a gel, a bead, plastics,glass, methacrylate, anionic particles, cationic particles, polarparticles, nonpolar particles, hydrophobic particles, hydrophilicparticles, ligand exchange particles, ion-pairing particles, sizeexclusion particles, or affinity chromatography particles.
 6. Theapparatus of claim 1, wherein the chromatography particles are adsorbentparticles.
 7. The apparatus of claim 6, wherein the fluidizedchromatography bed is configured to separate the heterogeneous liquid byadsorbing the target molecules to the adsorbent particles.
 8. Theapparatus of claim 6, wherein the fluidized chromatography bed isconfigured to separate the heterogeneous liquid by allowing materialsthat are not the target molecule to pass through the chromatography bed.9. The apparatus of claim 6, wherein the heterogeneous liquid comprisesa purified feed stock, tissue extract, chemical reaction mixture, stockbroth, bacterial homogenate, bacterial lysate, E. coli inclusion bodies,products secreted from bacteria, yeast, insect, animal, or plant cells,or tissue homogenates of bacteria, yeast, insect, animal, or plantcells, yeast cell homogenates, cellular homogenates, whole hybridomafermentation broth, myeloma cell culture, whole animal cell culturebroth, milk, animal tissue extracts, plant tissue extracts, unknownsource materials, chemical reaction mixtures, metal slurries, or culturesupernatant from a continuous fluidized bed bioreactor.